Assessment Of Peripheral Nerve Regeneration Research Articles

Effect of mesenchymal stem cells and melatonin on experimentally induced peripheral nerve injury in rats

Injury of a peripheral nerve (PNI) leads to both ischemic and inflammatory alterations. Sciatic nerve injury (SNI) represents the most widely used model for PNI. Mesenchymal stem cell-based therapy (MSCs) has convenient properties on PNI by stimulating the nerve regeneration. Melatonin has cytoprotective activity. The neuroprotective characteristics of MSCs and melatonin separately or in combination remain a knowledge need. In the rats-challenged SNI, therapeutic roles of intralesional MSCs and intraperitoneal melatonin injections were evaluated by functional assessment of peripheral nerve regeneration by walking track analysis involving sciatic function index (SFI) and two electrophysiological tests, electromyography and nerve conduction velocity, as well as measurement of antioxidant markers in serum, total antioxidant capacity (TAC) and malondialdehyde, and mRNA expression of brain derived neurotrophic factor (BDNF) in nerve tissues in addition to the histopathological evaluation of nerve tissue. Both individual and combination therapy with MSCs and melatonin therapies could effectively ameliorate this SNI and promote its regeneration as evidenced by improving the SFI and two electrophysiological tests and remarkable elevation of TAC with decline in lipid peroxidation and upregulation of BDNF levels. All of these led to functional improvement of the damaged nerve tissues and good recovery of the histopathological sections of sciatic nerve tissues suggesting multifactorial synergistic approach of the concurrent usage of melatonin and MSCs in PNI. The combination regimen has the most synergistic neuro-beneficial effects in PNI that should be used as therapeutic option in patients with PNI to boost their quality of life.

Criteria for assessing peripheral nerve injury. Behavioral and functional assessment in non-operated Wistar rats.

PurposeTo evaluate the normality pattern in functional tests of peripheral nerves.MethodsSixty female and sixty male Wistar rats were submitted to vibrissae movement and nictitating reflex for facial nerve; grooming test and grasping test for brachial plexus; and walking tracking test and horizontal ladder test for lumbar plexus. The tests were performed separately, with an interval of seven days between each.ResultsAll animals showed the best score in vibrissae movement, nictitating reflex, grooming test, and horizontal ladder test. The best score was acquired for the first time in more than 90% of animals. The mean of strength on the grasping test was 133.46±12.08g for the right and 121.74±8.73g for the left anterior paw. There was a difference between the right and left sides. There was no difference between the groups according to sex. There is no statistical difference comparing all functional indexes between sex, independent of the side analyzed. The peroneal functional index showed higher levels than the sciatic and tibial functional index on both sides and sex.ConclusionsThe behavioral and functional assessment of peripheral nerve regeneration are low-cost, easy to perform, and reliable tests. However, they need to be performed by experienced researchers to avoid misinterpretation.

Three-dimensional and non-destructive characterization of nerves inside conduits using laboratory-based micro computed tomography

BackgroundHistological assessment of peripheral nerve regeneration in animals is tedious, time-consuming and challenging for three-dimensional analysis. New methodThe present study reports on how and to what extent micro computed tomography of paraffin-embedded samples can provide a reliable three-dimensional approach for quantitative analysis of peripheral nerves. ResultsRat sciatic nerves were harvested, formalin-fixated, positioned into nerve conduits (NC), paraffin-embedded, and imaged using a laboratory-based X-ray microtomography system with an isotropic voxel length of 4μm. Suitable quantitative measures were identified and automatically evaluated, i.e. nerve length, cross-sectional area and volume, as well as vascular structures, to be used as an assessment and comparison indicator of regeneration quality. Comparison with existing methodsCompared to imaging using contrast agents, the investigated specimens can subsequently undergo the conventional histological analysis without requiring additional preparation steps. Contrast and spatial resolution are also increased significantly. ConclusionsWe demonstrate the potential of the micro computed tomography for non-destructive monitoring of peripheral nerves inside the conduits.

Functional recovery of sciatic nerve through inside-out vein graft in rats

Objective Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model. Methods The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery. Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and Immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments. Results Functional analysis of myelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves. Conclusion Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis

In vivo assessment of peripheral nerve regeneration by diffusion tensor imaging

To evaluate the sensitivity of diffusion tensor imaging (DTI) in assessing peripheral nerve regeneration in vivo. We assessed the changes in the DTI parameters and histological analyses after nerve injury to examine degeneration and regeneration in the rat sciatic nerves. For magnetic resonance imaging (MRI), 16 rats were randomly divided into two groups: group P (permanently crushed; n = 7) and group T (temporally crushed; n = 9). Serial MRI of the right leg was performed before the operation, and then performed at the timepoints of 1, 2, 3, and 4 weeks after the crush injury. The changes in fractional anisotropy (FA), axial diffusivity (λ(∥)), and radial diffusivity (λ(⟂)) were quantified. For histological analyses, the number of axons and the myelinated axon areas were quantified. Decreased FA and increased λ(⟂) were observed in the degenerative phase, and increased FA and decreased λ(⟂) were observed in the regenerative phase. The changes in FA and λ(⟂) were strongly correlated with histological changes, including axonal and myelin regeneration. DTI parameters, especially λ(⟂) , can be good indicators for peripheral nerve regeneration and can be applied as noninvasive diagnostic tools for a variety of neurological diseases.

Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration

Accurate assessment of peripheral nerve regeneration requires fixation techniques that preserve tissue in a natural state with minimal artifact. While transcardial perfusion fixation is accepted as the gold standard for tissue fixation, the less cumbersome approach of immersion fixation has been criticized for introducing artifacts in brain tissue. We investigated whether immersion fixation increased artifact compared to perfusion fixation in the rat sciatic nerve. Eighteen Lewis rats were randomized into three groups: glutaraldehyde immersion fixation; glutaraldehyde transcardial perfusion; and paraformaldehyde transcardial perfusion. All animals underwent sciatic nerve transection and repair followed by tissue harvest and fixation at three weeks. Qualitative assessment of neural architecture and histological features was followed by quantitative analysis of nerve regeneration parameters. Outcome measures included quantitative histomorphometry, analysis of axon/myelin ratios, assessment of fiber distributions, and ultrastructural analysis. No qualitative or quantitative differences were observed with immersion fixation when compared to the transcardial perfusion fixation methods. Immersion fixation is a valid method for assessment of peripheral nerve regeneration in a rat model.

Evaluation Methods in the Assessment of Peripheral Nerve Regeneration

Evaluation Methods in the Assessment of Peripheral Nerve Regeneration

Employment of the mouse median nerve model for the experimental assessment of peripheral nerve regeneration

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.

The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: a systematic review

Object A number of evaluation methods that are currently used to compare peripheral nerve regeneration with alternative repair methods and to judge the outcome of a new paradigm were hypothesized to lack resolving power. This would too often lead to the conclusion that the outcome of a new paradigm could not be discerned from the outcome of the current gold standard, the autograft. As a consequence, the new paradigm would incorrectly be judged as successful. Methods An overview of the methods that were used to evaluate peripheral nerve regeneration after grafting of the rat sciatic nerve was prepared. All articles that were published between January 1975 and December 2004 and concerned grafting of the rat sciatic nerve (minimum graft length 5 mm) and in which the experimental method was compared with an untreated or another grafted nerve were included. The author scored the presence of statistically significant differences between paradigms. Results Evaluation of nerve fiber count, nerve fiber density, N-ratio, nerve histological success ratio, compound muscle action potential, muscle weight, and muscle tetanic force are methods that were demonstrated to have resolving power. Conclusions A number of evaluation methods are not suitable to demonstrate a significant difference between experimental paradigms in peripheral nerve regeneration. It is preferable to apply a combination of evaluation methods with resolving power to evaluate nerve regeneration properly.

The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: a systematic review

A number of evaluation methods that are currently used to compare peripheral nerve regeneration with alternative repair methods and to judge the outcome of a new paradigm were hypothesized to lack resolving power. This would too often lead to the conclusion that the outcome of a new paradigm could not be discerned from the outcome of the current gold standard, the autograft. As a consequence, the new paradigm would incorrectly be judged as successful. An overview of the methods that were used to evaluate peripheral nerve regeneration after grafting of the rat sciatic nerve was prepared. All articles that were published between January 1975 and December 2004 and concerned grafting of the rat sciatic nerve (minimum graft length 5 mm) and in which the experimental method was compared with an untreated or another grafted nerve were included. The author scored the presence of statistically significant differences between paradigms. Evaluation of nerve fiber count, nerve fiber density, N-ratio, nerve histological success ratio, compound muscle action potential, muscle weight, and muscle tetanic force are methods that were demonstrated to have resolving power. A number of evaluation methods are not suitable to demonstrate a significant difference between experimental paradigms in peripheral nerve regeneration. It is preferable to apply a combination of evaluation methods with resolving power to evaluate nerve regeneration properly.

Recovery of neurophysiological features with time after rat sciatic nerve repair: a magneto‐neurographic study

Experimental assessment of peripheral nerve regeneration in rats by electrophysiology is controversial due to low reproducibility of electrophysiological indicators and diminished quantitative evaluation in conventional experimental set-ups. Magnetoneurography (MNG) counteracts these drawbacks by magnetically recording electrophysiological signals ex vivo, thereby providing accurate and quantitative data. In 50 rats, sciatic nerve transection was followed by direct repair. MNG outcome parameters, footprints [static toe spread factor (TSF); function] and muscle weight (MW) were studied for their recovery pattern from 2 to 24 weeks. By using MNG, we showed that the regeneration process still continues when functional recovery (static TSF) becomes stagnant. With regression analysis, MNG parameters amplitude, amplitude area and conduction velocity (CV) demonstrated moderate significant correlation with MW, whereas CV was not significantly associated with static TSF. No significant association exists between MW and static TSF. A Kaplan-Meier survival curve revealed that autotomy/contracture of rat hind paws was not related to decreased MNG outcome values. In conclusion, this study highlights and discusses the dissimilarities between direct (MNG) and indirect (static TSF and MW) assessment techniques of the regeneration process. We emphasise the significance of MNG as a direct derivative of axon regeneration in experimental rat studies. Additionally, we stress the must for right-left ratios, as neurophysiological indicators vary with age, and we confute possible bias in footprint analysis caused by exclusion of autotomy/contracture animals.

Static footprint analysis: a time‐saving functional evaluation of nerve repair in rats

Walking track analysis is a widely accepted technique for functional evaluation after sciatic nerve repair in rats, but it is labour-intensive. In 2000, Bervar described a time-saving digitised static footprint analysis. In that study there were good correlations between the traditional sciatic function index (SFI) and the newly-developed static sciatic index (SSI) and static toe spread factor (TSF), respectively. Despite promising results, static footprint analysis is still not widely used. The present study was designed to validate it. After transection of the sciatic nerve, end-to-end repair was assessed using video recorded dynamic and static footprints in 45 Wistar rats. We found an even better correlation between the SFI and both the SSI and the static TSF. In conclusion, static footprint analysis is a time-saving and easy technique for accurate functional assessment of peripheral nerve regeneration in rats.

The grasping test: a simple behavioral method for objective quantitative assessment of peripheral nerve regeneration in the rat

Behavioral recovery is the major goal of neuroscience research on the peripheral nervous system. However, behavioral methods of assessing peripheral nerve regeneration are still lacking. In the present work we used the median nerve and finger flexors as the experimental model. After resection or crush injury of the median nerve, rats were submitted daily to the grasping test. For this, they were gently lifted by the tail and allowed to grasp a grid connected to an ordinary electronic balance. While grasping the animal continued to be lifted by the tail with increasing firmeness until it lost its grip. At this precise moment the value shown by the balance was recorded. In the crushed median nerve, recovery of function was clearly demonstrated by the grasping test. The test also indicated the exact day on which recovery began and its improvement with time. This very simple objective behavioral method provides a sensitive quantitative technique for assessing recovery. The experimental model using the median nerve seems to be better than the sciatic nerve model, as no articular contractures or autotomies were noted in the present experimental animals.